The brain is composed of a great assortment of functionally diverse neurons connected in circuits. A greater understanding of neuronal diversity and the role of defined neuronal subtypes in neural circuits will enhance our fundamental understanding of the brain and foster progress in the treatment of neurological and mental health disorders. A major obstacle is a lack of tools for characterizing neuronal subtypes and manipulating them in vivo to assess their function Neuronal identity is specified by the expression of genes that give rise to specific neuronal properties, which, in turn, determine neuronal function within connected circuits. Beyond gene expression, the subcellular compartmentalization of neuronal proteins is a critical component of functional connectivity. Thus, determining the precise cellular and subcellular expression patterns of the proteins that underlie connectivity, such as neurotransmitter receptors, ion channels and cell adhesion molecules, will substantially advance our understanding of the molecular and biochemical logic of neural circuits. Here, we propose experiments to optimize CRISPR/Cas9-mediated HDR in Drosophila (Aim 1) and develop an innovative CRISPR/Cas9- based toolkit to simultaneously tag the endogenous genes that underlie neuronal connectivity (Aim 2) and gain experimental control of the neurons that express the targeted gene (Aim 3).